Dimethyl 7-[ω-N-(phthalimido)alkyl]aminonaphthalene-1,2-dicarboxylates

ABSTRACT

-Dimethyl 7-[ω-N-(phthalimido)alkyl]aminonaphthalene -1,2-dicarboxylates of the formula: ##STR1## wherein R is hydrogen or straight chain alkyl containing 1-4 carbon atoms and n=2-6. The compounds are intermediates in the synthesis of chemiluminescent naphthalene-1,2-dicarboxylic acid hydrazide-labeled conjugates which are useful as reagents in specific binding assays for determining ligands or their specific binding partners in liquid media.

This is a division of application Ser. No. 927,286, filed July 24, 1978.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel chemiluminescent-labeled conjugates foruse in specific binding assays for a ligand, such as an antigen, haptenor antibody, in a liquid medium such as a body fluid. The inventionfurther relates to intermediate compounds produced in the synthesis ofthe novel labeled conjugates.

2. Brief Description of the Prior Art

Specific binding assay methods have undergone a technological evolutionfrom the original competitive binding radioimmunoassay (RIA) in which aradioisotope-labeled antigen is made to compete with antigen from a testsample for binding to specific antibody. In the RIA technique, sampleantigen is quantitated by measuring the proportion of radioactivitywhich becomes associated with the antibody by binding of theradio-labeled antigen (the bound-species of the labeled antigen) to theradioactivity that remains unassociated from antibody (the free-species)and then comparing that proportion to a standard curve. A comprehensivereview of the RIA technique is provided by Skelly et al, Clin. Chem.29:146(1973). While by definition RIA is based on the binding ofspecific antibody with an antigen or hapten, radiolabeled binding assayshave been developed based on other specific binding interactions, suchas between hormones and their binding proteins.

From the radiolabeled binding assays have evolved nonradioisotopicbinding assays employing labeling substances such as enzymes asdescribed in U.S. Pat. Nos. 3,654,090 and 3,817,837. Recently furtherimproved nonradioisotopic binding assays have been developed asdescribed in German Offenlegungschriften Nos. 2,618,419 and 2,618,511based on U.S. Ser. Nos. 667,982 and 667,996, filed on Mar. 18, 1976 andassigned to the present assignee, employing particularly unique labelingsubstances, including coenzymes, cyclic reactants, cleavable fluorescentenzyme substrates, and chemiluminescent molecules. The chemiluminescentlabels consist of an organic molecule which undergoes a change inchemical structure with the production of light.

Specific examples of substances useful as chemiluminescent labelsmentioned in German OLS No. 2,618,511 are luminol, isoluminol,pyrogallol and luciferin. In particular, an example is provided in theOLS [and in Anal. Chem. 48:1933(1976) based on the same work] of anisoluminol-labeled conjugate wherein isoluminol is coupled through itsamino function by a 2-hydroxypropylene bridge to the ligand biotin. Theisoluminol-labeled conjugate is monitored in the binding assay bymeasuring the production of light in the presence of either hydrogenperoxide and peroxidase or potassium superoxide. The chemiluminescentphthalhydrazide-labeled conjugates wherein an amino-phthalhydrazide iscoupled through its amino function by a 2-hydroxyalkylene bridge to aligand are described in the U.S. patent application filed on even dateherewith entitled "Chemiluminescent-Labeled Conjugates for Use inSpecific Binding Assays" (U.S. Ser. No. 927,622) and assigned to thepresent assignee.

The efficiency of the amino-phthalhydrazides as chemiluminescent labelshas been improved by coupling through the amino function with a straightchain lower alkylene bridge as described in the U.S. patent applicationfiled on even date herewith entitled "ChemiluminescentPhthalhydrazide-Labeled Conjugates" (U.S. Ser. No. 927,621) and assignedto the present assignee. The use of more efficient labels enables moresensitive detection of ligands.

SUMMARY OF THE INVENTION

Labeled conjugates comprising even more efficient chemiluminescentlabels have now been devised having the formula: ##STR2## wherein R ishydrogen or straight chain alkyl containing 1-4 carbon atoms, preferablyethyl, n=2-6, preferably 4, and L(CO-- is a specifically bindableligand, or a binding analog thereof, bound through an amide bond.

The subject chemiluminescent naphthalene-1,2-dicarboxylic acidhydrazide-labeled conjugates are used in specific binding assays fordetecting the ligand or a binding partner thereof. The labeledconjugates are monitored in the performance of a binding assay byoxidizing the labeled conjugates and measuring the light produced eitheras total light produced or peak light intensity. For instance, aspecific binding assay for determining a hapten in a liquid medium mightbe carried out by incubating a sample of the liquid medium with anantibody for such hapten and with a labeled conjugate of the presentinvention wherein such hapten or a binding analog is labeled with thesubject chemiluminescent moiety. During the incubation, any haptenpresent in the liquid medium competes with the labeled conjugate forbinding with the antibody. Thereafter, the amount of labeled conjugateresulting in the bound-species compared to the free-species (whichamount is an inverse function of the amount of hapten in the liquidmedium assayed) is determined (i.e., monitored) either in a homogeneousfashion, if the chemiluminescent character of the labeled conjugate isdifferent when in the bound-species than when in the free-species, or ina heterogeneous fashion, if such character is essentially the same inboth species. In the homogeneous assay, the unseparated reaction mixturecontaining both species of the labeled conjugate is combined with anappropriate oxidation system for the chemiluminescent label and thelight produced is measured. In the heterogeneous assay, the bound- andfree-species are separated by any conventional technique, the oxidationsystem combined with one thereof, and the light produced is measured.

The monitorable chemiluminescent reaction may be illustrated as follows:##STR3## wherein hν represents electromagnetic radiation emitted. Usefuloxidation systems include hydrogen peroxide combined with any of thefollowing catalysts, peroxidase (particularly microperoxidase),catalase, deuterohemin, hematin or ferricyanide ions; hypochlorite ionscombined with cobalt ions; persulfate ions; potassium superoxide;periodate ions; hypoxanthine combined with xanthine oxidase; orpotassium t-butoxide.

The chemiluminescent-labeled conjugates may be employed in anyconventional homogeneous or heterogeneous binding assay method,including competitive binding methods, sequential saturation methods,direct binding methods, and "sandwich" binding methods. Further detailsconcerning the state of the art for binding assay techniques may befound in the aforementioned German OLS Nos. 2,618,419 and 2,618,511.

In the context of this disclosure, the following terms shall be definedas follows unless otherwise indicated: "specifically bindable ligand" isan organic substance of analytical interest for which there is aspecific binding partner; "specific binding partner of the ligand" isthe substance which has a noncovalent binding affinity for the ligand tothe exclusion of other substances; and "binding analog of the ligand" isan organic substance which is different in chemical structure from theligand but which behaves essentially the same as the ligand with respectto the binding affinity of the specific binding partner of the ligand.

The specifically bindable ligand or analog thereof in the presentlabeled conjugates, in terms of its chemical nature, usually is aprotein, polypeptide, peptide, carbohydrate, glycoprotein, steroid, orother organic molecule for which a specific binding partner isobtainable. In functional terms, the ligand will usually be an antigenor an antibody thereto; a hapten or an antibody thereto; or a hormone,vitamin, or drug, or a receptor or binding substance therefor. Mostcommonly, the ligand is an immunologically-active polypeptide or proteinof molecular weight between 1,000 and 4,000,000 such as an antigenicpolypeptide or protein or an antibody; or is a hapten of molecularweight between 100 and 1,500.

The present labeled conjugates are prepared usually by forming a peptideor amide couple between (1) an amino derivative of a chemiluminescentnaphthalene-1,2-dicarboxylic acid hydrazide and (2) either the ligand,where such contains a carboxylic acid function, or a binding analog ofthe ligand (e.g., a derivative of the ligand) which analog contains thedesired carboxylic acid function. Such condensation reactions can beaccomplished by reacting the amino derivatives of the label directlywith the carboxylic acid-containing ligand or ligand analog usingconventional peptide condensation reactions such as the carbodiimidereaction [Science 144:1344 (1964)], the mixed anhydride reaction[Erlanger et al, Methods in Immunology and Immunochemistry, ed. Williamsand Chase, Academic Press (New York 1967) p. 149], and the acid azideand active ester reactions [Kopple, Peptides and Amino Acids, W. A.Benjamin, Inc. (New York 1966)]. See also for a general review Clin.Chem. 22:726(1976).

It will be recognized, of course, that other well known methods areavailable for coupling the ligand or a derivative thereof to theamino-derivative of the label. In particular, conventional bifunctionalcoupling agents may be employed for coupling a ligand, or itsderivative, containing a carboxylic acid or amino group to theamino-derivative of the label. For example, amine-amine coupling agentssuch as bis-isocyanates, bis-imidoesters and glutaraldehyde [Immunochem.6: 53(1969)] may be used to couple a ligand or derivative containing anamino group to the amino-derivative of the label. Also, appropriatecoupling reactions are well known for inserting a bridge group incoupling an amine (e.g., the amino-derivative of the label) to acarboxylic acid (e.g., the ligand or a derivative thereof). Couplingreactions of this type are thoroughly discussed in the literature, forinstance in the above-mentioned Kopple monograph and in Lowe & Dean,Affinity Chromatography, John Wiley & Sons (New York 1974).

Such coupling techniques will be considered equivalents to thepreviously discussed peptide condensation reactions in preparing usefullabeled conjugates. The choice of coupling technique will depend on thefunctionalities available in the ligand or analog thereof for couplingto the label derivative and on the length of bridging group desired. Inall cases, for purposes of this disclosure, the resulting labeledconjugate will comprise the label derivative bound to the remainingportion of the conjugate through an amide bond. Such remaining portionof the conjugate will be considered as a residue of a binding analog ofthe ligand, unless the ligand itself is directly coupled to the labelderivative. Thus, in this description and in the claims to follow, theabbreviation L(CO-- represents the ligand or a binding analog thereofcoupled through an amide bond, wherein such analog may be a derivativeof the ligand coupled by peptide condensation to the label derivative ormay be the ligand or derivative thereof coupled through a bridging groupinserted by coupling of the ligand or derivative to the label derivativewith a bifunctional coupling agent.

Preparation of the present chemiluminescent-labeled conjugates proceedsaccording to the following general synthetic sequence: ##STR4##

Reaction of an N-(ω-bromoalkyl)phthalimide (I) [available from AldrichChemical Co., Milwaukee, Wis. USA, or see Derscherl and Weingarten,Justus Liebig's Annalen der Chemie 574:131(1951)] with dimethyl7-aminonaphthalene-1,2-dicarboxylate [Gundermann et al, Justus Liebig'sAnnalen der Chemie 684:127(1965)] produces the intermediate dimethyl7-[ω-N-(phthalimido)alkyl]aminonaphthalene-1,2-dicarboxylate (II).##STR5##

Alkylation of the amine group in the intermediate dicarboxylate (II) isobtained by reaction with a dialkyl sulfate (III) [Rodd, Chemistry ofCarbon Compounds, vol. 1, Elsevier Publ. Co. (New York 1951) p. 337]##STR6## to yield the alkylated derivative (IV) ##STR7## wherein R' isstraight chain alkyl containing 1-4 carbon atoms.

Treatment of the intermediate dicarboxylate (II) or the alkylatedderivative (IV) with hydrazine produces the amino-hydrazide (V) ##STR8##wherein R is hydrogen or straight chain alkyl containing 1-4 carbonatoms.

Condensation of the amino-hydrazide (V) with (a) the ligand to belabeled, where such contains a carboxylic acid function, (b) a bindinganalog of the ligand, such analog being a carboxylic acid derivative ofthe ligand, or (c) the ligand or an appropriate derivative of the ligandin the presence of a bifunctional coupling agent, produces thechemiluminescent-labeled conjugate (VI) ##STR9## wherein R is the sameas defined above and L(CO-- represents the specifically bindable ligand,or a binding analog thereof (formed by derivation of the ligand and/orinsertion of a bridge by a bifunctional coupling agent), bound throughan amide bond.

Other variations of labeled conjugates based on the above-describedsynthetic scheme are clearly evident. In particular, variousring-substituted dimethyl 7-aminonaphthalene-1,2-dicarboxylates may beused as starting material to produce ring-substituted labeled conjugatespossessing substantially the same qualitative properties as theconjugates prepared according to the above-described scheme. Suchconjugates will be recognized as equivalents and are exemplified by theaddition of one, two or more simple substituents to an availablearomatic ring site, such substituents including without limitation,alkyl, e.g., methyl, ethyl and butyl; halo, e.g., chloro and bromo;nitro; hydroxyl; alkoxy, e.g., methoxy and ethoxy, and so forth.

As stated hereinabove, the ligand which is comprised in the labeledconjugate or whose binding analog is comprised in the labeled conjugateis in most circumstances an immunologically-active polypeptide orprotein of molecular weight between 1,000 and 4,000,000 such as anantigenic polypeptide or protein or an antibody; or is a hapten ofmolecular weight between 100 and 1,500. Following will now be presentedvarious methods for coupling such ligands or analogs thereof to theamino-derivative (V) of the label through an amide bond.

Polypeptides and Proteins

Representative of specifically bindable protein ligands are antibodiesin general, particularly those of the IgG, IgE, IgM and IgA classes, forexample hapatitis B antibodies; and antigenic proteins such as insulin,chorionic gonadotropin (e.g., HCG), carcinoembryonic antigen (CEA),myoglobin, hemoglobin, follicle stimulating hormone, human growthhormone, thyroid stimulating hormone (TSH), human placental lactogen,thyroxine binding globulin (TBG), intrinsic factor, transcobalamin,enzymes such as alkaline phosphatase and lactic dehydrogenase, andhepatitis-associated antigens such as hepatitis B surface antigen (HB₂Ag), hepatitis e antigen (HB_(e) Ag) and hepatitis core antigen (HB_(c)Ag). Representative of polypeptide ligands are angiotensins I and II,C-peptide, oxytocin, vasopressin, neurophysin, gastrin, secretin, andglucagon.

Since, as peptides, ligands of this general category comprise numerousavailable carboxylic acid and amino groups, coupling to theamino-derivative of the chemiluminescent label can proceed according toconventional peptide condensation reactions such the carbodiimidereaction, the mixed anhydride reaction, and so forth as describedhereinabove, or by the use of conventional bifunctional reagents capableof coupling carboxylic acid or amino functions to the amino group in thelabel derivative as likewise described above. General referencesconcerning the coupling of proteins to primary amines or carboxylicacids are mentioned in detail above.

Haptens

Haptens, as a class, offer a wide variety of organic substances whichevoke an immunochemical response in a host animal only when injected inthe form of an immunogen conjugate comprising the hapten coupled to acarrier molecule, almost always a protein such as albumin. The couplingreactions for forming the immunogen conjugates are well developed in theart and in general comprise the coupling of a carboxylic acid ligand ora carboxylic acid derivative of the ligand to available amino groups onthe protein carrier by formation of an amide bond. Such well knowncoupling reactions are directly analogous to the present formation oflabeled conjugates by coupling carboxylic acid ligands or bindinganalogs to the amino-derivative of the chemiluminescent label.

Hapten ligands which themselves contain carboxylic acid functions, andwhich thereby can be coupled directly to the amino-derivative of thelabel, include the iodothyronine hormones such as thyroxine andliothyronine, as well as other materials such as biotin, valproic acid,folic acid and certain prostaglandins. Following are representativesynthetic routes for preparing carboxylic acid binding analogs of haptenligands which themselves do not contain an available carboxylic acidfunction whereby such analogs can be coupled to the amino-derivative ofthe label by the aforementioned peptide condensation reactions orbifunctional coupling agent reactions (in the structural formulae below,n represents an integer, usually from 1 through 6).

Carbamazepine

Dibenz[b,f]azepine is treated sequentially with phosgene, anω-aminoalkanol, and Jones reagent (chromium trioxide in sulfuric acid)according to the method of Singh, U.S. Pat. No. 4,058,511 to yield thefollowing series of carboxylic acids: ##STR10##

Quinidine

Following the method of Cook et al, Pharmacologist 17: 219(1975),quinidine is demethylated and treated with 5-bromovalerate followed byacid hydrolysis to yield a suitable carboxylic acid derivative.

Digoxin and Digitoxin

The aglycone of the cardiac glycoside is treated with succinic anhydrideand pyridine according to the method of Oliver et al, J. Clin. Invest.47:1035(1968) to yield the following: ##STR11##

Theophylline

Following the method of Cook et al, Res. Comm. Chem. Path. Pharm.13:497(1976), 4,5-diamino-1,3-dimethylpyrimidine-2,6-dione is heatedwith glutaric anhydride to yield the following: ##STR12##

Phenobarbital and Primidone

Sodium phenobarbital is heated with methyl 5-bromovalerate and theproduct hydrolyzed to the corresponding acid derivative of phenobarbital[Cook et al, Quantitative Analytic Studies in Epilepsy, ed. Kelleway andPeterson, Raven Press (New York 1976) pp. 39-58]: ##STR13##

To obtain the acid derivative of primidone following the same Cook et alreference method, 2-thiophenobarbital is alkylated, hydrolyzed, and theproduct treated with Raney nickel to yield: ##STR14##

Diphenylhydantoin

Following the method of Cook et al, Res. Comm. Chem. Path. Pharm.5:767(1973), sodium diphenylhydantoin is reacted with methyl5-bromovalerate followed by acid hydrolysis to yield the following:##STR15##

Morphine

Morphine free base is treated with sodium β-chloroacetate according tothe method of Spector et al, Science 168:1347 (1970) to yield a suitablecarboxylic acid derivative.

Nicotine

According to the method of Langone et al, Biochem. 12(24): 5025(1973),trans-hydroxymethylnicotine and succinic anhydride are reacted to yieldthe following: ##STR16##

Androgens

Suitable carboxylic acid derivatives of testosterone and androstenedionelinked through either the 1- or 7-position on the steroid nucleus areprepared according to the method of Bauminger et al, J. Steroid Biochem.5:739(1974). Following are representative testosterone derivatives:##STR17##

Estrogens

Suitable carboxylic acid derivatives of estrogens, e.g., estrone,estradiol and estriol, are prepared according to the method of Baumingeret al, supra, as represented by the following estrone derivative:##STR18##

Progesterones

Suitable carboxylic acid derivatives of progesterone and its metaboliteslinked through any of the 3-, 6- or 7-positions on the steroid nucleusare prepared according to the method of Bauminger et al, supra, asrepresented by the following progesterone derivatives: ##STR19##

The methods described above are but examples of the many knowntechniques for forming suitable carboxylic acid derivatives of haptensof analytical interest. The principal derivation techniques arediscussed in Clin. Chem. 22:726(1976) and include esterification of aprimary alcohol with succinic anhydride [Abraham and Grover, Principlesof Competitive Protein-Binding Assays, ed. Odell and Daughaday, J. B.Lippincott Co. (Philadelphia 1971) pp. 140-157], formation of an oximefrom reaction of a ketone group with carboxylmethyl hydroxylamine [J.Biol. Chem. 234:1090(1959)], introduction of a carboxyl group into aphenolic residue using chloroacetate [Science 168:1347(1970)], andcoupling to diazotized p-aminobenzoic acid in the manner described in J.Biol. Chem. 235:1051(1960).

The hereinbefore-described general synthetic sequence for preparing thepresent chemiluminescent-labeled conjugates is specifically exemplifiedby the following description of the preparation of the labeled thyroxineconjugate7-[N-ethyl-N-(4-thyroxinylamido)butyl]aminonaphthalene-1,2-dicarboxylicacid hydrazide. The reaction sequence for this synthesis is outlined inTable 1 which follows.

A. Preparation of the Labeled Conjugate Dimethyl7-[4-N-(Phthalimido)butyl]aminonaphthalene-1,2-dicarboxylate (3)

A solution of 14.1 grams (g) (0.05 mol) of N-(4-bromobutyl)phthalimide(1) (Aldrich Chemical Co., Milwaukee, Wis. USA) and 25.0 g (0.1 mol) ofdimethyl 7-aminonaphthalene-1,2-dicarboxylate (2) [Gundermann et al,Justus Liebig's Annalen der Chemie 684:127(1965)] in 100 milliliters(ml) of 2,2,2-trifluoroethanol was refluxed under argon for 16 hours.Evaporation gave a residue that was partitioned between 400 ml of etherand 300 ml of water (H₂ O). The ether phase was separated, dried, andevaporated. The resulting dark red residue was chromatographed on acolumn of 1200 g of silica gel (E. Merck, Darmstadt, West Germany)eluting with a 19:1 volume to volume (v:v) mixture of benzene andmethanol. After the first 700 ml of eluent was discarded, 20 mlfractions

                                      TABLE 1                                     __________________________________________________________________________     ##STR20##                                                                     ##STR21##                                                                     ##STR22##                                                                     ##STR23##                                                                     ##STR24##                                                                     ##STR25##                                                                     ##STR26##                                                                     ##STR27##                                                                     ##STR28##                                                                     ##STR29##                                                                     ##STR30##                                                                    __________________________________________________________________________

were collected. Fractions numbered 219 to 251 were combined andevaporated to give 9 g of the substituted carboxylate (3) as a clear redoil.

Analysis:

NMR Spectrum (CDCl₃): δ1.7 (m, 4H), 3.9 (s, 3H), 4.0 (s, 3H).

Infrared Spectrum (CDCl₃): 1720 cm⁻¹ (carbonyl).

Mass Spectrum (70 eV) m/e: 461 [MH⁺ ], 460 [M⁺ ], 429 [M⁺ minus OCH₃ ].

Dimethyl7-{N-Ethyl-N-[4-(N-phthalimido)butyl]amino}naphthalene-1,2-dicarboxylate(4)

A mixture of 9 g (0.02 mol) of the substituted carboxylate (3) and 20 mlof diethyl sulfate was heated at 130° C. for two hours under argon. Thedark solution was poured into a beaker of crushed ice containing 200 mlof saturated aqueous sodium bicarbonate solution. When all the ice hadmelted, the mixture was extracted with three 250 ml volumes of ether.The ether extracts were combined, dried, and evaporated to give a redoil. The oil was chromatographed on a column of 600 g of silica geleluting with a 19:1 (v:v) mixture of benzene and methanol and the lightyellow fractions combined and evaporated. Excess diethyl sulfate wasremoved by evaporative sublimation at 50° C. and reduced pressure of0.01 millimeters (mm) mercury leaving a residue of 4 g of the N-ethylsubstituted carboxylate (4) as a light red oil.

Analysis:

NMR Spectrum (CDCl₃): δ3.9 (s, 3H), 4.1 (s, 3H)

Mass Spectrum (70 eV) m/e: 489 [MH⁺ ], 488 [M⁺ ], 457 [M⁺ minus OCH₃ ]

7-[N-(4-Aminobutyl)-N-ethyl]aminonaphthalene-1,2-dicarboxylic AcidHydrazide (5)

A mixture of 4 g (0.008 mol) of the N-ethyl substituted carboxylate (4),15 ml of 85% hydrazine, and 50 ml of methanol was refluxed for threehours. When cool, the mixture was evaporated to dryness on a rotaryevaporator and the crystalline residue scraped out and dried overnightat 80° C. under high vacuum. The resulting dark solid waschromatographed on a column of 200 g of silica gel (E. Merck, Darmstadt,West Germany), eluting with a 7:3 (v:v) mixture of ethanol and 1 molar(M) triethylammonium bicarbonate and collecting 20 ml fractions.Fractions numbered 25 to 75 were combined and evaporated to give ayellow solid. After two recrystallizations from pyridine, there wascollected 1.1 g of the amino-hydrazide (5) as give yellow crystals,melting point (m.p.) 246°-247° C.

Analysis: Calculated for C₁₈ H₂₂ N₄ O₂ : C, 66.24; H, 6.80; N, 17.17.Found: C, 66.05; H, 6.69; N, 17.65

NMR Spectrum (d₆ -DMSO): δ1.1 (m, 3H), 1.5 (m, 4H).

Infrared Spectrum (KCl): 1615 cm⁻¹ (carbonyl).

The efficiency of the amino-derivative (5) of the label in achemiluminescent reaction and the detection limit of such derivativewere determined as follows.

In determining efficiency, the label derivative and luminol(5-amino-2,3-dihydrophthalazine-1,4-dione) were oxidized individually atseveral levels in the picomolar range and related to the peak lightintensities by a graph plot. Linear portions of the resulting curvesallowed calculation of change in peak light intensity per unitconcentration for the label derivative and for luminol. Efficiency ofthe label derivative was expressed as a percentage of the slope producedwith luminol.

Reaction mixtures (150 μl) of the following composition were assembledin 6×50 mm test tubes mounted in a Dupont 760 Luminescence Biometer (E.I. duPontde Nemours and Co., Wilmington, Delaware USA) with asensitivity setting of 820: 50 mM sodium hydroxide, 57.5 mM barbitaladjusted to pH 8.6, 0.27 μM microperoxidase (Sigma Chemical Co., St.Louis, Missouri USA) and either the amino-derivative of the label orluminol at varying concentrations in the picomolar (pM) range (dilutedwith H₂ O from a stock solution at 1 mM in 0.1 M soium carbonate, pH10.5). The final pH of the reaction mixtures was 12.6. Each mixture wasincubated 10 minutes at room temperature and 10 μl of 90 mM hydrogenperoxide in 10 mM Tris-HCl buffer [tris-(hydroxymethyl)-aminomethanehydrochloride], pH 7.4 was added to initiate the chemiluminescentreaction. Peak light intensity values were recorded from the instrumentreadings. All reactions were performed in triplicate and averaged. Theefficiency of the label derivative (5) was found to be 420%.

Detection limit was defined as the concentration of the label derivativethat produced a peak light intensity one and a half times the backgroundchemiluminescence in the reaction mixture. The detection limit for thelabel derivative (5) was found to be 0.1 pM.

N-Trifluoroacetylthyroxine (6)

A solution of 20 g [25.6 millimoles (mmol)] of L-thyroxine (SigmaChemical Co., St. Louis, Missouri USA) in 240 ml of dry ethyl acetatecontaining 46 ml of trifluoroacetic acid and 7.6 ml of trifluoroaceticanhydride was stirred at 0° C. for one hour. Upon warming to roomtemperature and adding 200 ml of H₂ O, a suspension formed which wasthen saturated with sodium chloride. The organic phase of the mixturewas separated, washed with saturated aqueous sodium chloride solution,dried over anhydrous magnesium sulfate, filtered and evaporated to give21.3 g of the N-protected thyroxine derivative (6). A sample wasrecrystallized from ether-pentane to give fine crystals, m.p. 233°-235°C. (decomposed).

Analysis: Calculated for C₁₇ H₁₀ F₃ I₄ NO₅ : C, 23.39; H, 1.15; N, 1.60.Found: C, 23.23; H, 1.12; N, 1.56.

Infrared Spectrum (KCl): 1700 cm⁻¹ (carbonyl).

Optical Rotation [α]_(D) ²⁵ =-14.97° (c 1.0, dimethylsulfoxide). 4

7-[N-Ethyl-N-(4-thyroxinylamido)butyl]aminonaphthalene-1,2-dicarboxylicAcid Hydrazide (7)

A solution of 1.746 g (2 mmol) of N-trifluoroacetylthyroxine (6) in 20ml of dry pyridine was cooled to -10° C. with stirring under argon. Tothis solution was added 450 milligrams (mg) (2.2 mmol) of dicyclohexylcarbodiimide, followed 45 minutes later by 980 mg (3 mmol) of theamino-hydrazide (5). After stirring for 3 hours at -10° C., the reactionwas allowed to warm to room temperature overnight. The reaction mixturewas diluted with 10 ml of pyridine, 10 g of silica gel was added, andthe solvent removed under vacuum. The resulting iumpregnated silica gelwas placed atop a 200 g column of silica gel made up in a 7:3 (v:v)mixture of ethanol and 1 M triethylammonium bicarbonate, eluting withthe same solvent mixture. After the first 400 ml of eluent wasdiscarded, 20 ml fractions were collected. Fractions numbered 19 to 30were combined and evaporated to give a yellow crystalline residue. Thisproduct was refluxed for 3 hours in 300 ml of 1 M triethylammoniumbicarbonate to complete removal of the trifluoroacetyl protecting group.The solution was then filtered while hot. When cool, 15 g of silica gelwas added and the solvent evaporated. The impregnated silica gel wasplaced atop a 200 g column of silica gel and eluted with a 7:3 (v:v)mixture of ethanol and 1 M triethylammonium bicarbonate, collecting 20ml fractions. Fractions numbered 30 to 50 were combined and evaporatedto give 910 mg of the labeled thyroxine conjugate (7) as a yellow solid.A 110 mg sample was chromatographed on a 45×3.2 centimeter (cm) columnof Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden), elutingwith methanol. Fractions of 7 ml volume were collected and thosenumbered 64 to 76 were combined and evaporated to give 60 mg of thelabeled conjugate (7) as a yellow solid, m.p. 218° C. (decomposed).

Analysis: Calculated for C₃₃ H₃₁ I₄ N₅ O₅ : C, 36.52; H, 2.88; I, 46.77;N, 6.45. Found: C, 35.61; H, 3.02; I, 44.69; N, 6.25.

B. Binding Assay for Thyroxine

Competitive binding reaction mixtures (200 82 l) were assembled intriplicate by combining the following reagents: 50 μl of 10 nM labeledconjugate (7) in 75 mM bactital buffer (pH 8.6), 50 μl of a preparationof antibody to thyroxine in the same buffer, varying volumes of 54.8 nMthyroxine in the same buffer, and a sufficient volume of the buffer tomake a final volume of 200 μl. After a 10 minute incubation at roomtemperature, the free- and bound-species of the labeled conjugate wereseparated for each reaction mixture by applying a 150 μl aliquot tosmall Sephadex G-25 (Pharmacia Fine Chemicals, Uppsala, Sweden) columns.The columns had a bed volume of 1.5 ml and were pre-washed withsuccessive 3 ml volumes of 7% acetic acid (3 times), H₂ O 0.1 M sodiumhydroxide (3 times), and 75 mM of the barbital buffer. The bound-speciesof the labeled conjugate was eluted from the column with 1.5 ml of thebarbital buffer leaving the free-species in the column.

An aliquot (95 μl) of each column effluent was added to 55 μl of asolution of 134 mM sodium hydroxide, 0.73 μM microperoxidase, and 27 mMbarbital in a 6×50 mm test tube. After a 10 minute incubation at roomtemperature, each tube was placed in the Dupont 760 Biometer and 10 μlof 90 mM hydrogen peroxide in 10 mM Tris-HCl buffer (pH 7.4) was added.The resulting peak intensity of the light produced in thechemiluminescent reaction was recorded from the instrument reading. Eachbinding mixture was monitored in triplicate to give a total of 9individual peak light intensity values which were averaged for eachdifferent volume of thyroxine added to the initial reaction mixture.

The peak intensity values were also related as a percentage of totallabeled conjugate in the bound-species by ratioing such values to thepeak light intensity measured in the chemiluminescent reaction using 95μl of 0.25 nM labeled conjugate in place of the column effluent.Background chemiluminescence in the monitoring reaction was found to be0.3 peak intensity units.

The relationships of the amount of thyroxine in the binding reaction topeak light intensity and percent of labeled conjugate in thebound-species are shown in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        volume of thyroxine                                                                          peak light    percent in                                       solution added (μl)                                                                       intensity     bound-species                                    ______________________________________                                         0             26.5          63.1                                              5             25.3          59.5                                             10             23.3          55.5                                             20             23.7          56.4                                             40             19.1          45.4                                             60             14.7          35.0                                             80             13.7          32.6                                             ______________________________________                                    

The results demonstrate that the labeled conjugate of the presentinvention is useful in binding assays for determining a ligand in aliquid medium.

What is claimed is:
 1. A compound of the formula: ##STR31## wherein R ishydrogen or straight chain alkyl containing 1-4 carbon atoms and n=2-6.2. The compound of claim 1 wherein n=4.
 3. The compound of claim 2wherein R is ethyl.